Multiply-substituted protease variants

ABSTRACT

Novel enzyme variants including protease variants derived from the DNA sequences of naturally-occurring or recombinant non-human proteases are disclosed. The variant proteases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant protease to generate the substitution of a plurality of amino acid residues in the amino acid sequence of a precursor protease. Such variant proteases have properties which are different from those of the precursor protease, such as altered wash performance. The substituted amino acid residue correspond to positions 27, 45, 170, 181, 251 and 271 of  Bacillus amyloliquefaciens  subtilisin. Additional variants comprising at least one additional substitution at a position selected from 1, 14, 49, 61, 87, 100, 102, 118, 128, 204 and 258 of  Bacillus amyloliquefaciens  subtilisin are also described.

This is a Divisional of co-pending U.S. patent application Ser. No.10/500,943, filed on Apr. 20, 2005, which is a 371 filing ofPCT/US03/01448, filed Jan. 16, 2003, which claims priority to U.S.Provisional Patent Application Ser. No. 60/350,222 filed Jan. 16, 2002.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of carbonyl hydrolases. They comprise adiverse class of enzymes having a wide range of specificities andbiological functions. Stroud, R. Sci. Amer., 131:74-88. Despite theirfunctional diversity, the catalytic machinery of serine proteases hasbeen approached by at least two genetically distinct families ofenzymes: 1) the subtilisins and 2) the mammalian chymotrypsin-relatedand homologous bacterial serine proteases (e.g., trypsin and S. gresiustrypsin). These two families of serine proteases show remarkably similarmechanisms of catalysis. Kraut, J. (1977), Annu. Rev. Biochem.,46:331-358. Furthermore, although the primary structure is unrelated,the tertiary structure of these two enzyme families bring together aconserved catalytic triad of amino acids consisting of serine, histidineand aspartate.

Subtilisins are serine proteases (approx. MW 27,500) which are secretedin large amounts from a wide variety of Bacillus species and othermicroorganisms. The protein sequence of subtilisin has been determinedfrom at least nine different species of Bacillus. Markland, F S., et al.(1983), Hoppe-Seyler'Z. Physiol. Chem., 364:1537-1540. Thethree-dimensional crystallographic structure of subtilisins fromBacillus amyloliquefaciens, Bacillus licheniformis and several naturalvariants of B. lentus have been reported. These studies indicate thatalthough subtilisin is genetically unrelated to the mammalian serineproteases, it has a similar active site structure. The x-ray crystalstructures of subtilisin containing covalently bound peptide inhibitors(Robertus, J. D., et al. (1972), Biochemistry, 11:2439-2449) or productcomplexes (Robertus, J. D., et al. (1976), J. Biol. Chem.,251:1097-1103) have also provided information regarding the active siteand putative substrate binding cleft of subtilisin. In addition, a largenumber of kinetic and chemical modification studies have been reportedfor subtilisin; Svendsen, B. (1976), Carlsberg Res. Commun., 41:237-291;Markland, F. S. Id.) as well as at least one report wherein the sidechain of methionine at residue 222 of subtilisin was converted byhydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al.(1965), J. Biol. Chem., 244:5333-5338) and extensive site-specificmutagenesis has been carried out (Wells and Estell (1988) TIBS13:291-297)

SUMMARY OF THE INVENTION

One aspect of the invention, the charge distribution of a molecule isaltered to affect its orientation and interaction with phases, surfaces,other molecules and fields.

An enzyme variant of a precursor or parent enzyme is contemplatedherein, the variant comprising one or more modifications at a chargedamino acid residue position, the variant being characterized by havingthe same net electrostatic charge and/or the same isoelectric point asthe precursor enzyme.

In another aspect of the present invention, a protease variant of aprecursor protease is contemplated herein, the variant comprising one ormore modifications at a charged amino acid residue position, the variantbeing characterized by having the same net electrostatic charge orisoelectric point as the precursor protease. The charged amino acids canbe aspartic acid, glutamic acid, histidine, lysine, tyrosine andarginine. The residue positions can be those equivalent to positions 5,7, 23, 26, 28-31, 34, 47, 63, 65, 66, 69, 70, 73, 82-85, 88, 90, 92, 93,105, 113, 125, 138, 139, 148-151, 176, 178, 179, 193, 196, 200, 201,202, 207, 219, 220, 223, 229, 233, 250, 266, 267 and 273 of Bacillusamyloliquefaciens subtilisin are identified herein. The residuepositions can also be those equivalent to positions 27, 39, 41, 45, 67,94, 136, 170, 181, 247, 251 and/or 271 of Bacillus amyloliquefacienssubtilisin. It is a further aspect to provide DNA sequences encodingsuch protease variants, as well as expression vectors containing suchvariant DNA sequences.

A protease variant of a precursor protease, said variant comprising oneor more modifications at a charged amino acid residue position, saidvariant being characterized by having the same net electrostatic chargeas said precursor protease. The protease variant of claim 1, whereinsaid charged amino acid is selected from the group consisting ofaspartic acid, glutamic acid, lysine and arginine. The protease variantcomprises an amino acid sequence having a substitution at one or moreresidue positions equivalent to residue positions selected from thegroup consisting of 27, 45, 170, 181, 251 and 271 of Bacillusamyloliquefaciens subtilisin as set forth in SEQ ID NO. 2. The proteasevariant comprising a substitution at one or more positions correspondingto 27, 45, 170, 181, 251 and 271 is a substitution selected from K27T,R45N, R170S, D181N, K251G and E271T.

The protease variant may further comprise an additional substitution atone or more positions corresponding to 1, 14, 49, 61, 87, 100, 102, 118,128, 204 and 258 of Bacillus amyloliquefaciens subtilisin as set forthin SEQ ID NO.2. Variants can be selected from the combinations ofR45N-G118E-E271R, R45N-P14R, R45N-N₂O₄R, D181N-G118D, R45N-G258R,R170S-A1R, R170S-G61R, R170S-N204R, K251G-S87K, R170S-S216R,E271T-G100E, E271T-G102E, E271T-S128E, K27T-G100E, R170S-G100R,E271T-S49E and E271T-S128E.

Still further, another aspect of the invention is to provide host cellstransformed with such vectors.

There is further provided a cleaning composition comprising a proteasevariant of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C depict the DNA (SEQ ID NO: 1) and amino acid sequences (SEQID NO:2) for Bacillus amyloliquefaciens subtilisin and a partialrestriction map of this gene.

FIG. 2 depicts the conserved amino acid residues among subtilisins fromBacillus amyloliquefaciens (BPN)′ and Bacillus lentus (wild-type).

FIGS. 3A and 3B depict the amino acid sequence of four subtilisins. Thetop line represents the amino acid sequence of subtilisin from Bacillusamyloliquefaciens subtilisin (also sometimes referred to as subtilisinBPN′) (SEQ ID NO:3). The second line depicts the amino acid sequence ofsubtilisin from Bacillus subtilis (SEQ ID NO:4). The third line depictsthe amino acid sequence of subtilisin from B. licheniformis (SEQ IDNO:5). The fourth line depicts the amino acid sequence of subtilisinfrom Bacillus lentus (also referred to as subtilisin 309 in PCTWO89/06276) (SEQ ID NO:6). The symbol * denotes the absence of specificamino acid residues as compared to subtilisin BPN′.

FIG. 4 depicts the pVS08 B. subtillis expression vector.

FIG. 5 depicts the orientation of the forward ApaI primer, the reverseApaI primer, the reverse mutagenic primer, and the forward mutagenicprimer.

DETAILED DESCRIPTION OF THE INVENTION

Proteases are carbonyl hydrolases which generally act to cleave peptidebonds of proteins or peptides. As used herein, “protease” means anaturally-occurring protease or a recombinant protease.Naturally-occurring proteases include α-aminoacylpeptide hydrolase,peptidylamino acid hydrolase, acylamino hydrolase, serinecarboxypeptidase, metallocarboxypeptidase, thiol proteinase,carboxyl-proteinase and metalloproteinase. Serine, metallo, thiol andacid proteases are included, as well as endo and exo-proteases.

The present invention includes protease enzymes which are non-naturallyoccurring carbonyl hydrolase variants (protease variants) having adifferent proteolytic activity, stability, substrate specificity, pHprofile and/or performance characteristic as compared to the precursorcarbonyl hydrolase from which the amino acid sequence of the variant isderived. Specifically, such protease variants have an amino acidsequence not found in nature, which is derived by substitution of aplurality of amino acid residues of a precursor protease with differentamino acids. The precursor protease may be a naturally-occurringprotease or a recombinant protease.

The protease variants useful herein encompass the substitution of any ofthe nineteen naturally occurring L-amino acids at the designated aminoacid residue positions. Such substitutions can be made in any precursorsubtilisin (procaryotic, eucaryotic, mammalian, etc.). Throughout thisapplication reference is made to various amino acids by way of commonone- and three-letter codes. Such codes are identified in Dale, M. W.(1989), Molecular Genetics of Bacteria, John Wiley & Sons, Ltd, AppendixB.

The protease variants useful herein are preferably derived from aBacillus subtilisin. More preferably, the protease variants are derivedfrom Bacillus lentus subtilisin and/or subtilisin 309.

Subtilisins are bacterial or fungal proteases which generally act tocleave a peptide bonds of proteins or peptides. As used herein,“subtilisin” means a naturally-occurring subtilisin or a recombinantsubtilisin. A series of naturally-occurring subtilisins is known to beproduced and often secreted by various microbial species. Amino acidsequences of the members of this series are not entirely homologous.However, the subtilisins in this series exhibit the same or similar typeof proteolytic activity. This class of serine proteases shares a commonamino acid sequence defining a catalytic triad which distinguishes themfrom the chymotrypsin related class of serine proteases. The subtilisinsand chymotrypsin related serine proteases both have a catalytic triadcomprising aspartate, histidine and serine. In the subtilisin relatedproteases the relative order of these amino acids, reading from theamino to carboxy terminus, is aspartate-histidine-serine. In thechymotrypsin related proteases, the relative order, however, ishistidine-aspartate-serine. Thus, subtilisin herein refers to a serineprotease having the catalytic triad of subtilisin related proteases.Examples include but are not limited to the subtilisins identified inFIG. 3 herein. Generally and for purposes of the present invention,numbering of the amino acids in proteases corresponds to the numbersassigned to the mature Bacillus amyloliquefaciens subtilisin sequencepresented in FIG. 1.

“Recombinant subtilisin” or “recombinant protease” refer to a subtilisinor protease in which the DNA sequence encoding the subtilisin orprotease is modified to produce a variant (or mutant) DNA sequence whichencodes the substitution, deletion or insertion of one or more aminoacids in the naturally-occurring amino acid sequence. Suitable methodsto produce such modification, and which may be combined with thosedisclosed herein, include those disclosed in U.S. Pat. No. RE 34,606,U.S. Pat. No. 5,204,015 and U.S. Pat. No. 5,185,258, U.S. Pat. No.5,700,676, U.S. Pat. No. 5,801,038, and U.S. Pat. No. 5,763,257.

“Non-human subtilisins” and the DNA encoding them may be obtained frommany procaryotic and eucaryotic organisms. Suitable examples ofprocaryotic organisms include gram negative organisms such as E. coli orPseudomonas and gram positive bacteria such as Micrococcus or Bacillus.Examples of eucaryotic organisms from which subtilisin and their genesmay be obtained include yeast such as Saccharomyces cerevisiae, fungisuch as Aspergillus sp.

An “enzyme variant” has an amino acid sequence which is derived from theamino acid sequence of a “precursor enzyme”. The precursor enzymesproteases include naturally-occurring enzymes and recombinant enzymes.Enzymes contemplated by the inventors include, but are not limited tooxidoreductases, transferases, hydrolases, lyases, isomerases, andligases. Specific exemplary enzymes contemplated by the inventorsinclude, but are not limited to amylases, laccases, proteases,dehydrogenases, and permeases. The amino acid sequence of the enzymevariant is “derived” from the precursor enzyme amino acid sequence bythe substitution, deletion or insertion of one or more amino acids ofthe precursor amino acid sequence. Such modification is of the“precursor enzyme DNA sequence” which encodes the amino acid sequence ofthe precursor enzyme rather than manipulation of the precursor enzymeper se. Suitable methods for such manipulation of the precursor DNAsequence include methods disclosed herein, as well as methods known tothose skilled in the art. It is contemplated that any reference ordiscussion regarding proteases may be applicable to other enzymes, e.g,those identified in part above,

A “protease variant” has an amino acid sequence which is derived fromthe amino acid sequence of a “precursor protease” The precursorproteases include naturally-occurring proteases and recombinantproteases. The amino acid sequence of the protease variant is “derived”from the precursor protease amino acid sequence by the substitution,deletion or insertion of one or more amino acids of the precursor aminoacid sequence. Such modification is of the “precursor DNA sequence”which encodes the amino acid sequence of the precursor protease ratherthan manipulation of the precursor protease enzyme per se. Suitablemethods for such manipulation of the precursor DNA sequence includemethods disclosed herein, as well as methods known to those skilled inthe art (see, for example, EP 0 328299, WO89/06279 and the US patentsand applications already referenced herein).

“Charged amino acid” is defined as an amino acid that is potentiallyionizable, changes charge and provides an electrostatic charge at aspecified pH or pH range. These amino acids include, for example, acidicamino acids, basic amino acids and some polar amino acids. Acidic aminoacids are those that are negatively charged at pH 6.0, for exampleaspartic acid (Asp or D) and/or glutamic acid (Glu or E). Basic aminoacids are those that are positively charged at pH 6.0, for examplelysine (Lys or K), arginine (Arg or R), and/or Histidine (H is or H).

“Uncharged amino acid” is defined as an amino acid that is notpotentially ionizable. These amino acids include, but are not limited touncharged nonpolar amino acids and/or uncharged polar amino acids.Uncharged nonpolar amino acids include, but are not limited to alanine(Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile orI), proline (Pro or P), phenylalanine (Phe or F), tryptophan (Trp or W),and/or methionine (Met or M). Uncharged polar amino acids include, butare not limited to glycine (Gly or G), serine (Ser or S), threonine (Thror T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N)and/or glutamine (Gln or Q).

“Net electrostatic charge” is defined as the sum of the electrostaticcharges of the variant or precursor enzyme or protease at a given pH orpH range. An exemplary pH is pH 6.0.

“Isoelectric Point” (pl_(o)) is defined as the pH value where theprotein or protein complex, e.g., the protease or protease complex (withoptionally attached metal or other ions) is neutral, i.e. the sum ofelectrostatic charges (net electrostatic charge=NEC) on the complex isequal to zero. In this sum consideration of the positive or negativenature of the individual electrostatic charges must be taken intoaccount.

The “same isoelectric point (pl_(o)) is defined as the pl_(o) beingwithin a defined range of pH units. For example, a defined range of pHunits could be no greater than 1 pH unit, between 0.25 and 0.75, forexample 0.5 pH units, preferably within 0.01 and 0.5 pH units, forexample 0.1 pH units, and more preferably within 0.001 and 0.05 pHunits, for example 0.01 pH units of the pl_(o) to which the other pl_(o)is being compared. The same isoelectric point can be determined at agiven pH or at a defined pH range.

“Identical electrostatic charge” is defined as maintaining “Z” or thesame number of specific charged residues in the protease variant asthere are in the precursor protease. While the number of the chargedresidues may be the same, the specific amino acid residues may besubstituted into other positions about the exterior of the proteasevariant so long as the net electrostatic charge is the same as that ofthe precursor protease at a given pH.

The “same net electrostatic charge” is defined as maintaining Z, or thesum of the electrostatic charges of the protease precursor within adefined range of the sum of electrostatic charges of the proteasevariant. Maintaining the same sum of electrostatic charge means keepingthe net electrostatic charge within a defined range of charge units overa defined range of pH. A protease variant having the same netelectrostatic charge as the protease precursor would have a netelectrostatic charge within a defined number of charge units of theprecursor protease. For example the protease variant having the same netelectrostatic charge can be no greater than 1 pH unit within 0.25 to0.75 charge units, e.g., 0.5 units, of the precursor protease netelectrostatic charge. Still more preferably the protease variant havingthe same net electrostatic charge can be within 0.05 to 0.25 units,e.g., 0.1 units of the precursor protease net electrostatic charge.Still more preferably the protease variant having the same netelectrostatic charge can be within 0.001 to 0.05$ units, e.g., 0.01units of the precursor protease net electrostatic charge. Charge unitscan be defined as the number of protons donated (acid) or accepted(basic). The electrostatic charge of an individual amino acid can begenerally ascertained by determining the number of protons accepted ordonated at a given pH, for example 6.0 or 7.0. Z values can also bedetermined by the equations described later in this application.

“Total charge content” of the variant or precursor protease is definedas the total number of electrostatic charges of the respective protease.A protease variant with the same net electrostatic charge may have adifferent total charge content as the precursor protease.

As recognized by those of skill in the art, the isoelectric point can beconveniently calculated by using equilibrium considerations using pKvalues for the various charged residues in the enzyme in question andthen finding by iteration the pH value where the NEC of the enzymemolecule is equal to zero as described in EP 0945 502 and the examplestherein, which is expressly incorporated by reference herein.

One problem with this calculation is that the pK values for the chargedresidues are dependent on their environment and consequently subject tovariation. However, very good results are obtainable by allocatingspecific approximate pK values to the charged residues independently ofthe actual value. It is also possible to perform more sophisticatedcalculations, partly taking the environment into consideration.

The pl_(o) may also be determined experimentally by isoelectric focusingor by titrating a solution containing the enzyme. In addition, thevarious pK values for is the charged residues may be determinedexperimentally by titration.

In a further aspect of the invention, the above observations about thepl_(o) are further utilized in a method for determining or selecting theposition(s) and the amino acid(s) to be deleted, substituted or insertedfor the amino acid(s) in the precursor protease, so that the netelectrostatic charge or isoelectric point of the variant protease is thesame as the NEC or the pl_(o) of the precursor protease calculated atthe same pH value or a defined pH range.

Another way of expressing this principle covered by the invention isthat the position(s) and the amino acid(s) to be deleted, substituted orinserted for the amino acid(s) in said precursor protease or enzyme isselected in a way whereby the total number of charges or total chargecontent (=TCC) and/or the NEC in a resulting variant protease or enzymeis kept constant to provide for a variant protease or enzyme having anisoelectric point kept the same at a defined pH or pH range for optimumwash performance of the protease or enzyme, which pH optimum should beas close as possible to the pH of the wash liquor, wherein said mutantprotease is intended for use.

As indicated above, the pl_(o) of a macromolecule such as an enzyme iscalculated as the pH where the NEC of the molecule is zero. Theprocedure is exemplified in the examples described in EP 0 945 502, butthe principles are described in more detail here.

pK values are assigned to each potentially charged amino acid residue.Then the ratio of the occurrence of an amino acid residue at a given pHin charged or uncharged form (charged/uncharged, C/U(i)) is calculatedfor both negative and positive charges by using formulas Ia and Ib:

C/U(i)=10^((pH-pKi))(negative charge)  (Ia)

C/U(i)=10^((pKi-pH))(positive charge)  (Ib).

According to the above formulas, if pH equals pK_(i), C/U(i) is equalto 1. The relative charge, Q_(r)(i), or charge contribution allocated toeach charged residue is then calculated by using formulas IIa and IIb:

Q _(r)(i)=C/U(i)(1+C/U(i))(negative charge)  (IIa)

Q _(r)(i)=−C/U(i)(1+C/U(i))(positive charge)  (IIb)

The pH value where the sum of all the charge contributions from thecharged residues is equal to zero is can be found by iteration orthrough interpolation in a sufficiently dense pH-charge sum table.

Those skilled in the art will recognize that another method ofdetermining the net electrostatic charge Z as, if the group (such as theR or amino group) has a cationic acid form, α represents the fractionalpositive charge:

$Z = {{+ \alpha} = \frac{+ 1}{1 + 10^{({{pH} \cdot {pKa}})}}}$

On the other hand, for groups such as the carboxyl, with a neutral acidform and an anionic conjugate base, α represents the fraction uncharged.The fractional charge is then:

$Z = {{- \left( {1 - \alpha} \right)} = {{\alpha - 1} = \frac{- 1}{1 + 10^{({{pKa} - {pH}})}}}}$

It has been noted that various bulk protease or enzyme properties aredependent upon the NEC and/or isoelectric point of the protease orenzyme molecule. For example, the protease or enzyme solubility,stability, phase distribution in multiple phase media and/or surfacecharge are properties that are affected by an alteration of themolecule's NEC and/or isoelectric point. Surprisingly, improved proteasecharacteristics can be effected while maintaining the same isoelectricpoint or same net electrostatic charge. While not desiring to be boundby a particular theory, it is believed by the inventors that there aresituations where it is desirous to maintain the bulk properties of theprotein, enzyme, or protease in question while modifying the kinetics ofthe interaction of the molecule, e.g. the distribution of charges ororientation of the molecule relative a substrate, surface or media.

In one aspect of the invention, the protease variant and the precursorprotease have an identical net electrostatic charge or identicalisoelectric point. The same net electrostatic charge can be maintainedby having the identical electrostatic charge or compensating for thecharge change resulting from the additionally modified positions byadditional modifications to the amino acid sequence of the precursorprotease. These additional modifications include, but are not limited tosubstituting or inserting a residue that has an opposite charge to thatadditional residue (adding an additional acidic residue to compensatefor an additional basic residue). Thus it is contemplated that thenumber of charged residues in the protease variant may be different fromthat of the protease precursor, for example, when the number of chargedresidues is greater than in the protease precursor. To compensate forthe additional charged residue, a correspondingly oppositely chargedamino acid substitution can be made to maintain the same netelectrostatic charge. Additionally, the number of charged residues inthe protease or enzyme variant may be less than the number of chargedresidues in the protease or enzyme precursor if a correspondinglyoppositely charged amino acid deletion or substitution of anotheruncharged residue when a charged residue is deleted or substituted withan uncharged residue.

In one aspect of the present invention, is when the NEC or pl_(o) isidentical, for example when the total charge content of the proteasevariant and the precursor protease are the same, or when the same numberof charged residues in the precursor protease is maintained. When thecharged amino acid is repositioned to maintain the identical NEC orpl_(o), at least one of these charged amino acids is substituted into adifferent residue position from that of the precursor protease. If thereare a specified number of a specific charged amino acid in the proteaseprecursor, “X” lysines in Bacillus lentus (GG36), then the variantprotease will retain the same number of lysine residues, i.e., “X”, butat different positions relative to the precursor protease. Thus, forexample, K27 can be substituted with a different residue and thecorresponding K substituted at another position, preferably a surfaceposition. In one embodiment, a charged residue, e.g., glutamic acid,aspartic acid, lysine or arginine can be substituted into a differentposition. To maintain the identical electrostatic charge, the specificprecursor residue which is replaced by the charged residue, e.g., Kresidue from position 27, can be substituted in at position 27. Forexample an R45N-N204R has identical residue positions replaced tomaintain identical electrostatic charge. In addition, if the specificprecursor residue which is replaced by the charged residue is anuncharged residue, other uncharged residues can be substituted into aposition originally having a charged residue. For example an R170S-A1Rcombination replaces an alanine with an arginine while replacing anarginine with a serine. Of course, if multiple modifications are made,any replaced residue can be substituted into any of the other residuesbeing modified so long as the same number of each respective amino acidis maintained. The electrostatic charge can be determined at anypredetermined pH, so long as the determination is made at the same pHfor the protease variant and the precursor protease.

In another aspect of the invention, the same net electrostatic charge ofthe molecule is maintained by compensating for the change in netelectrostatic charge resulting from the modification to the precursorprotease. For example, one way to achieve such alteration is to insertor substitute in an additional oppositely charged amino acid residue ordelete a similarly charged but different amino acid residue. If, forexample, there are more acidic amino acids present in the proteasevariant than in the precursor protease, the variant will includeadditional basic amino acids. If, for example, there are more of aspecific acidic amino acid, for example, glutamic acid, present in theprotease variant than in the precursor protease, to compensate for suchmodifications, a corresponding number of aspartic acid residues could bedeleted or substituted with a non-charged amino acid. For example, ifthere is a specified number of a charged amino acids in the precursorprotease, the inventors contemplate increasing or decreasing the numberof that amino acid in the variant protease with a corresponding increaseor decrease in the amino acids that compensate for the change in thenumber of charged amino acids. Thus, as described above, additionalpositively charged residues could be compensated by the addition of acorresponding number of negatively charged amino acids or substitutionof a corresponding number of other positively charged amino acids with anon-charged residue or combinations thereof. A lesser number ofpositively charged residues could be compensated by the deletion of acorresponding number of negatively charged amino acids, substitution ofa corresponding number of non-charged residues for a correspondingnumber of negatively charged amino acids or combinations thereof.

In one embodiment, the same charged amino acid residue that is replacedby an uncharged residue at a first amino acid position is substitutedinto a second amino acid position where the same uncharged amino acidreplacing the charged residue is present. An uncharged residue can besubstituted at the original position of the charged amino acid, whilethe substituted charged amino acid can replace the position of theuncharged amino acid. For example, R45N-N204R reflects the substitutionof an uncharged amino acid, asparagine for a charged amino acid,arginine. The same uncharged amino acid substituted for the chargedamino acid need not be present at the position where the charged aminoacid is reinserted. For example, an uncharged amino acid selected fromthe group of alanine (Ala or A), glycine (Gly or G), asparagines (Asn orN), proline (Pro or P), serine (Ser or S) and/or threonine (Thr or T)can be substituted into the charged amino acid position while thecharged amino acid residue is substituted into an amino acid positionoriginally occupied by another in the above group. For example, in thevariant E271T-G100E, the glutamic acid amino acid at position 271 issubstituted with a threonine amino acid, while a glycine residue atposition 100 is replaced with a glutamic acid amino acid. Like wise, theidentical charged amino acid need not be substituted into the originallyuncharged position, e.g., K27T-G100E. The charged amino acids asparticacid (D), glutamic acid (E), lysine (K), and arginine (R) are useful inthis regard.

In still another aspect of the invention the protease variant NEC variesless than a range of 0.5 charge units (Z) from that of the precursorprotease NEC over the range of pH's from 0-14.

In still another aspect of the invention, the protease variant NECvaries less than a range of 1 charge unit from that of the precursorprotease NEC over a defined pH range. That defined pH range could be,for example within 2 pH units of the that recognized in the art as theoptimum or desired pH for the desired protease or enzyme environment orwithin a range of 4 pH units.

In another aspect of the invention, it has been determined that themodification of the charged residues found in the precursor proteasewhile maintaining the same or identical net electrostatic charge canresult in a protease variant displaying increased beneficial washcharacteristics.

In still another aspect of the invention, it has been determined thatthe modification of the charged residues found in the precursor proteasewhile maintaining the same or identical net electrostatic charge at adefined pH, over a defined pH range, e.g. over a range of 4 pH units, orover the range of pH from 0.001 to 14 can result in a protease variantdisplaying increased beneficial wash characteristics.

Exemplary charged amino acid residues contemplated for modification bythe inventors include, for example, basic amino acids such as lysine,arginine and/or histidine; acidic amino acids, for example aspartic acidand/or glutamic acid; and/or otherwise polar R groups, for exampletyrosine.

In another aspect, the variant proteases of the present invention have,relative to said precursor protease, the same number ofpositively-charged amino acid residue(s), both the identical amino acidsas in the precursor protease and different amino acids having the samecharge, and the same number of negatively-charged amino acid residue(s)as in the precursor protease; or either more or fewer positively-chargedamino acid residue(s) and a corresponding more or fewernegatively-charged amino acid residue(s), such that the netelectrostatic charge and/or the isoelectric point of the proteasevariant is the same as the precursor protease, while havingmodifications among the equivalent amino acid residues at any one ormore of positions: 5, 7, 22, 23, 24, 26, 28-31, 34, 45, 47, 63, 65, 66,69, 70, 73, 82-85, 88, 90, 92, 93, 97, 102, 105, 113, 125, 127, 138,139, 148-151, 169, 170, 176, 178, 179, 193, 196, 200, 201, 202, 207,219, 220, 223, 229, 233, 250, 266, 267 and 273 of Bacillusamyloliquefaciens (BPN′). In one embodiment, modifications among theequivalent amino acid residues at one or more of positions 27, 45, 136,170, 181, 247, 251 and/or 271 include the substitution of an unchargedresidue for a charged residue position. These residue positions are ofinterest since these equivalent positions in Bacillus lentus wild typehave charged amino acid residues at these positions. For example, theresidue positions at 27, 38, 40, 44, 65, 92, 134, 164, 175, 241, 245,and/or 265 of Bacillus lentus subtilisin (SEQ ID NO. 6) are equivalent,respectively, to 27, 39, 41, 45, 67, 94, 136, 170, 181, 247, 251 and/or271 of Bacillus amyloliquefacien (SEQ ID No. 2).

In another aspect, the variant proteases of the present invention have,relative to said precursor protease, the same number ofpositively-charged amino acid residue(s), both the identical amino acidsas in the precursor protease and different amino acids having the samecharge, and the same number of negatively-charged amino acid residue(s)as in the precursor protease; or either more or fewer positively-chargedamino acid residue(s) and a corresponding more or fewernegatively-charged amino acid residue(s), such that the netelectrostatic charge and/or the isoelectric point of the proteasevariant is the same as the precursor protease, while havingmodifications among the equivalent amino acid residues at any one ormore of positions: 27, 39, 41, 45, 67, 94, 136, 170, 181, 197, 247, 249,251, and 271 of Bacillus amyloliquefaciens (BPN′). Specificsubstitutions contemplated by the inventors include K27A, K27C, K27E,K27Q, K27G, K27H, K27I, K27L, K27M, K27F, K27P, K27S, K27T, K27W, K27Y,H39A, H39R, H39D, H39N, H39C, H39E, H39Q, H39G, H39H, H39I, H39L, H39K,H39M, H39F, H39P, H39T, H39W, H39Y, H39V, D41A, D41R, D41C, D41E, D41Q,D41G, D41H, D41I, D41L, D41K, D41M, D41F, D41P, D41S, D41T, D41W, D41Y,D41V, R45A, R45R, R45D, R45N, R45C, R45E, R45Q, R45G, R45H, R45I, R45L,R45K, R45M, R45F, R45P, R45S, R45T, R45W, R45Y, R45Vr H67A, H67R, H67D,H₆₇N, H₆₇C, H67E, H67Q, H67G, H67H, H67I, H67L, H67K, H67M, H₆₇F, H67P,H67S, H67T, H67W, H67Y, H67V, K94A, K94R, K94D, K94N, K94C, K94E, K94Q,K94G, K94H, K94I, K94L, K94K, K94M, K94F, K94P, K94S, K94T, K94W, K94Y,K94V, E136A, E136D, E136N, E136C, E136E, E136G, E136H, E136I, E136L,E136K, E136M, E136F, E136P, E136S, E136T, E136W, E136Y, E136V, R170A,R170R, R170D, R170N, R170C, R170E, R170Q, R170G, R170H, R170I, R170L,R170K, R170M, R170F, R170P, R170S, R170T, R170W, R170Y, R170V, D181A,D181R, D181D, D181N, D181C, D181E, D181Q, D181G, D181H, D181I, D181L,P181K, D131M, P181F, D181P, D181S, D181T, D181W, D181Y, D181V, D197A,D197R, D197D, D197N, D197C, D197E, D197Q, P197G, D197H, D197I, D197L,P197K, D197M, D197F, D197P, D197S, D197T, P197W, D197Y, P197V, R247A,R247R, R247D, R247N, R247C, R247E, R247Q, R247G, R247H, R247I, R247L,R247K, R247M, R247F, R247P, R247S, R247T, R247W, R247Y, R247V, H249A,H249R, H249D, H249N, H249C, H249E, H249Q, H249G, H249H, H249I, H249K,H249M, H249F, H249P, H249S, H249T, H249W, H249V, K251A, K251D, K251C,K251Q, K251G, K251H, K251I, K251L, K251K, K251M, K251F, K251P, K251S,K251T, K251W, K251Y, K251V, E271A, E271R, E271D, E271N, E271C, E271E,E271H, E271I, E271L, E271K, E271M, E271F, E271P, E271S, E271T, E271W,E271Y, and/or E271V of Bacillus amyloliquefaciens. It was noted that anincrease in the number of positive charged residues by substitutionthereof may result in an increase in the efficacy of that particularvariant in a particular wash environment, while a corresponding oppositecharge change could result in increased efficacy in a different washenvironment. For example it is anticipated that negative chargemutations provide beneficial characteristics in low ionic strength washenvironments and that positive charge mutations provide beneficialcharacteristics in high ionic strength wash environments. It isanticipated that variants that encompass both a positive increase and anegative increase while maintaining the same net electrostatic charge orisoelectric point will result in a protease molecule that exhibitsimproved characteristics in both environments as compared to theperformance of the precursor protease.

These substitutions are preferably made in Bacillus lentus (recombinantor native-type) subtilisin, although the substitutions may be made inany Bacillus protease, for example Bacillus amyloliquefaciens and/orSubtilisin 309.

One aspect of the present invention includes a protease variant furthercomprising at least one additional replaced amino acid at one or moreresidue positions equivalent to residue positions or selected from thegroup consisting of 1, 2-4, 6, 9-12, 14, 15, 17-20, 25, 27, 36-38, 40,44, 49, 51, 52, 54-61, 68, 71, 75, 76, 87, 89, 91, 97, 100-102, 104,108, 111, 112, 115, 117, 118, 120-123, 128, 129, 131, 133, 134, 136,137, 140, 143-146, 159, 164, 165, 167, 170, 171, 173, 175, 180, 182-187,191, 192, 194, 195, 204, 206, 209-212, 216, 218, 222, 224, 226, 234-245,252, 255, 257-263, 265, 268, 269, and 274. Specific substitutionscontemplated by the inventors include those equivalent to; I122A, Y195E,M222A, M222S, Y167A, R170S, A194P, D36, N76D, H120D, G195E, and K235N ofBacillus amyloliquefaciens or Bacillus lentus, which variant is derivedfrom a Bacillus subtilisin.

Of particular interest are variants at these positions demonstratingincreased wash performance with a charged amino acid substitution.Combination variants including these positions and those originallyhaving a charged amino acid are of interest. Exemplary combinationscontemplated by the inventors include K27T-G100E, R45N-A1R, R45N-P14R,R45N-G61R, R45N-S128R, R45N-N₂₀₄R, R45N-S216R, R45N-G258R, R170S-A1R,R170S-P14R, R170S-S49R, R170S-G61R, R170S-G100R, R170S-S128k,R170S-N₂₀₄R, R170S-S216R, R170S-G258R, D181N-G118D, D181N-G258D,K251G-S87K, E271T-S49E, E271T-T66E, E271T-G100E, E271T-G102E,E271T-S128E, R45N-G118E-E271R, S49R-G102E-R170S-E271T, andP14R-R45N-R170S-G258R. Those skilled in the art will recognize theprotease variants having these modifications can be made and aredescribed in U.S. Pat. Nos. 5,741,694; 6,190,900; and 6,197,567,expressly incorporated by reference herein. In addition, thesemodifications can also be made using direct Bacillus transformationmethods as described in Provisional Application Ser. No. 60/423,087(filed Nov. 1, 2002; Neelam Amin and Volker Schellenberger). In oneembodiment, the modifications were performed using fusion PCR techniques(Teplyakov, A V, et al, Protein Eng., 1992 Jul. 5(5):413-20).Provisional Application Ser. No. ______, filed concurrently this date(Chris Leeflang, et al.)

Still another aspect of the present invention includes a proteasevariant further comprising at least one additional replaced amino acidat one or more residue positions from the group consisting of 21, 22,24, 32, 33, 36, 50, 64, 67, 77, 87, 94, 95, 96, 97, 104, 107, 110, 124,123, 126, 127, 128, 129, 135, 152, 155, 157, 156, 166, 169, 170, 171,172, 189, 197, 204, 213, 214, 215, 217, 222, or 274 of Bacillusamyloliquefaciens. Specific residues contemplated by the inventorsinclude: K27R, M50F, N76D, S101G, S103A, V104I, V104Y, I122A, N123S,M124L, G159D, Y217L, A232V, Q236H, Q245R, N248D, N252K, T274A, andM222S. Protease variants, recombinant DNA encoding mutants at thesepositions and/or methods for making these modifications are described inU.S. Pat. Nos. RE 34,606, 5,972,682; 5,185,258; 5,310,675; 5,316,941;5,801,038; 5,972,682, 5,955,340 and 5,700,676, expressly incorporated byreference herein.

These amino acid position numbers refer to those assigned to the matureBacillus amyloliquefaciens subtilisin sequence presented in FIG. 1. Theinvention, however, is not limited to the mutation of this particularsubtilisin but extends to precursor proteases containing amino acidresidues at positions which are “equivalent” to the particularidentified residues in Bacillus amyloliquefaciens subtilisin. In apreferred embodiment of the present invention, the precursor protease isBacillus lentus subtilisin (SEQ ID NO. 6) and the substitutions are madeat the equivalent amino acid residue positions in B. lentuscorresponding to those listed above.

A residue (amino acid) position of a precursor protease is equivalent toa residue of Bacillus amyloliquefaciens subtilisin if it is eitherhomologous (i.e., corresponding in position in either primary ortertiary structure) or analogous to a specific residue or portion ofthat residue in Bacillus amyloliquefaciens subtilisin (i.e., having thesame or similar functional capacity to combine, react, or interactchemically).

In order to establish homology to primary structure, the amino acidsequence of a precursor protease is directly compared to the Bacillusamyloliquefaciens subtilisin primary sequence and particularly to a setof residues known to be invariant in subtilisins for which sequence isknown. For example, FIG. 2 herein shows the conserved residues asbetween B. amyloliquefaciens subtilisin and B. lentus subtilisin. Afteraligning the conserved residues, allowing for necessary insertions anddeletions in order to maintain alignment (i.e., avoiding the eliminationof conserved residues through arbitrary deletion and insertion), theresidues equivalent to particular amino acids in the primary sequence ofBacillus amyloliquefaciens subtilisin are defined. Alignment ofconserved residues preferably should conserve 100% of such residues.However, alignment of greater than 98%, greater than 95%, greater than90%, greater than 85%, greater than 80%, greater than 75%, greater than50% or at least greater than 45% of conserved residues is also adequateto define equivalent residues. Conservation of the catalytic triad,Asp32/His64/Ser221 should be maintained. Siezen et al. (1991) ProteinEng. 4(7):719-737 shows the alignment of a large number of serineproteases. Siezen et al. refer to the grouping as subtilases orsubtilisin-like serine proteases.

For example, in FIG. 3, the amino acid sequence of subtilisin fromBacillis amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis(carlsbergensis) and Bacillus lentus are aligned to provide the maximumamount of homology between amino acid sequences. A comparison of thesesequences shows that there are a number of conserved residues containedin each sequence. These conserved residues (as between BPN′ and B.lentus) are identified in FIG. 2.

These conserved residues, thus, may be used to define the correspondingequivalent amino acid residues of Bacillus amyloliquefaciens subtilisinin other subtilisins such as subtilisin from Bacillus lentus (PCTPublication No. WO89/06279 published Jul. 13, 1989), the preferredprotease precursor enzyme herein, or the subtilisin referred to as PB92(EP 0 328 299), which is highly homologous to the preferred Bacilluslentus subtilisin. The amino acid sequences of certain of thesesubtilisins are aligned in FIGS. 3A and 3B with the sequence of Bacillusamyloliquefaciens subtilisin to produce the maximum homology ofconserved residues. As can be seen, there are a number of deletions inthe sequence of Bacillus lentus as compared to Bacillusamyloliquefaciens subtilisin. Thus, for example, the equivalent aminoacid for Val 165 in Bacillus amyloliquefaciens subtilisin in the othersubtilisins is isoleucine for B. lentus and B. licheniformis.

“Equivalent residues” may also be defined by determining homology at thelevel of tertiary structure for a precursor protease whose tertiarystructure has been determined by x-ray crystallography. Equivalentresidues are defined as those for which the atomic coordinates of two ormore of the main chain atoms of a particular amino acid residue of theprecursor protease and Bacillus amyloliquefaciens subtilisin (N on N, CAon CA, C on C and O on) are within 0.13 nm and preferably 0.1 nm afteralignment. Alignment is achieved after the best model has been orientedand positioned to give the maximum overlap of atomic coordinates ofnon-hydrogen protein atoms of the protease in question to the Bacillusamyloliquefaciens subtilisin. The best model is the crystallographicmodel giving the lowest R factor for experimental diffraction data atthe highest resolution available.

${R\mspace{14mu} {factor}} = \frac{{\sum\limits_{h}{{{Fo}(h)}}} - {{{Fc}(h)}}}{\sum\limits_{h}{{{Fo}(h)}}}$

Equivalent residues which are functionally similar to a specific residueof Bacillus amyloliquefaciens subtilisin are defined as those aminoacids of the precursor protease which may adopt a conformation such thatthey either alter, modify or contribute to protein structure, substratebinding or catalysis in a manner defined and attributed to a specificresidue of the Bacillus amyloliquefaciens subtilisin. Further, they arethose residues of the precursor protease (for which a tertiary structurehas been obtained by x-ray crystallography) which occupy an analogousposition to the extent that, although the main chain atoms of the givenresidue may not satisfy the criteria of equivalence on the basis ofoccupying a homologous position, the atomic coordinates of at least twoof the side chain atoms of the residue lie with 0.13 nm of thecorresponding side chain atoms of Bacillus amyloliquefaciens subtilisin.The coordinates of the three dimensional structure of Bacillusamyloliquefaciens subtilisin are set forth in EP0 Publication No. 0 251446 (equivalent to U.S. Pat. No. 5,182,204, the disclosure of which isincorporated herein by reference) and can be used as outlined above todetermine equivalent residues on the level of tertiary is structure.

Some of the residues identified for substitution are conserved residueswhereas others are not. In the case of residues which are not conserved,the substitution of one or more amino acids is limited to substitutionswhich produce a variant which has an amino acid sequence that does notcorrespond to one found in nature. In the case of conserved residues,such substitutions should not result in a naturally-occurring sequence.The protease variants of the present invention include the mature formsof protease variants, as well as the pro- and prepro-forms of suchprotease variants. The prepro-forms are the preferred construction sincethis facilitates the expression, secretion and maturation of theprotease variants.

“Prosequence” refers to a sequence of amino acids bound to theN-terminal portion of the mature form of a protease which when removedresults in the appearance of the “mature” form of the protease. Manyproteolytic enzymes are found in nature as translational proenzymeproducts and, in the absence of post-translational processing, areexpressed in this fashion. A preferred prosequence for producingprotease variants is the putative prosequence of Bacillusamyloliquefaciens subtilisin, although other protease prosequences maybe used.

A “signal sequence” or “presequence” refers to any sequence of aminoacids bound to the N-terminal portion of a protease or to the N-terminalportion of a proprotease which may participate in the secretion of themature or pro forms of the protease. This definition of signal sequenceis a functional one, meant to include all those amino acid sequencesencoded by the N-terminal portion of the protease gene which participatein the effectuation of the secretion of protease under nativeconditions. The present invention utilizes such sequences to effect thesecretion of the protease variants as defined herein. One possiblesignal sequence comprises the first seven amino acid residues of thesignal sequence from Bacillus subtilis subtilisin fused to the remainderof the signal sequence of the subtilisin from Bacillus lentus (ATCC21536).

A “prepro” form of a protease variant consists of the mature form of theprotease having a prosequence operably linked to the amino terminus ofthe protease and a “pre” or “signal” sequence operably linked to theamino terminus of the prosequence.

“Expression vector” refers to a DNA construct containing a DNA sequencewhich is operably linked to a suitable control sequence capable ofeffecting the expression of said DNA in a suitable host. Such controlsequences include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites and sequences which controltermination of transcription and translation. The vector may be aplasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, “plasmid” and“vector” are sometimes used interchangeably as the plasmid is the mostcommonly used form of vector at present. However, the invention isintended to include such other forms of expression vectors which serveequivalent functions and which are, or become, known in the art.

The “host cells” used in the present invention generally are procaryoticor eucaryotic hosts which preferably have been manipulated by themethods disclosed in U.S. Pat. No. RE 34,606 and/or U.S. Pat. No.5,441,882 to render them incapable of secreting enzymatically activeendoprotease. A host cell useful for expressing protease is the Bacillusstrain BG2036 which is deficient in enzymatically active neutralprotease and alkaline protease (subtilisin). The construction of strainBG2036 is described in detail in U.S. Pat. No. 5,264,366. Other hostcells for expressing protease include Bacillus subtilis I168 (alsodescribed in U.S. Pat. No. RE 34,606, U.S. Pat. No. 5,441,882 and U.S.Pat. No. 5,264,366, the disclosure of which are incorporated herein byreference), as well as any suitable Bacillus strain such as B.licheniformis, B. lentus, etc, A particularly useful host cell is theBacillus strain BG2864. The construction of strain G2864 is described indetail in D. Naki, C. Paech, G. Ganshaw, V. Schellenberger. ApplMicrobiol Biotechnol (1998) 49:290-294.

Host cells are transformed or transfected with vectors constructed usingrecombinant DNA techniques. Such transformed host cells are capable ofeither replicating vectors encoding the protease variants or expressingthe desired protease variant. In the case of vectors which encode thepre- or prepro-form of the protease variant, such variants, whenexpressed, are typically secreted from the host cell into the host cellmedium.

“Operably linked,” when describing the relationship between two DNAregions, simply means that they are functionally related to each other.For example, a presequence is operably linked to a peptide if itfunctions as a signal sequence, participating in the secretion of themature form of the protein most probably involving cleavage of thesignal sequence. A promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; a ribosome binding siteis operably linked to a coding sequence if it is positioned so as topermit translation.

The genes encoding the naturally-occurring precursor protease may beobtained in accord with the general methods known to those skilled inthe art. The methods generally comprise synthesizing labeled probeshaving putative sequences encoding regions of the protease of interest,preparing genomic libraries from organisms expressing the protease, andscreening the libraries for the gene of interest by hybridization to theprobes. Positively hybridizing clones are then mapped and sequenced.

The cloned protease is then used to transform a host cell in order toexpress the protease. The protease gene is then ligated into a high copynumber plasmid. This plasmid replicates in hosts in the sense that itcontains the well-known elements necessary for plasmid replication: apromoter operably linked to the gene in question (which may be suppliedas the gene's own homologous promoter if it is recognized, i.e.,transcribed, by the host), a transcription termination andpolyadenylation region (necessary for stability of the mRNA transcribedby the host from the protease gene in certain eucaryotic host cells)which is exogenous or is supplied by the endogenous terminator region ofthe protease gene and, desirably, a selection gene such as an antibioticresistance gene that enables continuous cultural maintenance ofplasmid-infected host cells by growth in antibiotic-containing media.High copy number plasmids also contain an origin of replication for thehost, thereby enabling large numbers of plasmids to be generated in thecytoplasm without chromosomal limitations. However, it is within thescope herein to integrate multiple copies of the protease gene into hostgenome. This is facilitated by procaryotic and eucaryotic organismswhich are particularly susceptible to homologous recombination.

The gene can be a natural B. lentus gene. Alternatively, a syntheticgene encoding a naturally-occurring or mutant precursor protease may beproduced. In such an approach, the DNA and/or amino acid sequence of theprecursor protease is determined. Multiple, overlapping syntheticsingle-stranded DNA fragments are thereafter synthesized, which uponhybridization and ligation produce a synthetic DNA encoding theprecursor protease. An example of synthetic gene construction is setforth in Example 3 of U.S. Pat. No. 5,204,015, the disclosure of whichis incorporated herein by reference.

Once the naturally-occurring or synthetic precursor protease gene hasbeen cloned, a number of modifications are undertaken to enhance the useof the gene beyond synthesis of the naturally-occurring precursorprotease. Such modifications include the production of recombinantproteases as disclosed in U.S. Pat. Nos. RE 34,606; 5,741,694;6,190,900; 6,197,567; 5,972,682; 5,185,258; 5,700,676 and EPOPublication No. 0 251 446 and the production of protease variantsdescribed herein.

The following cassette mutagenesis method may be used to facilitate theconstruction of the protease variants of the present invention, althoughother methods may be used. First, the naturally-occurring gene encodingthe protease is obtained and sequenced in whole or in part. Then thesequence is scanned for a point at which it is desired to make amutation (deletion, insertion or substitution) of one or more aminoacids in the encoded enzyme. The sequences flanking this point areevaluated for the presence of restriction sites for replacing a shortsegment of the gene with an oligonucleotide pool which when expressedwill encode various mutants. Such restriction sites are preferablyunique sites within the protease gene so as to facilitate thereplacement of the gene segment. However, any convenient restrictionsite which is not overly redundant in the protease gene may be used,provided the gene fragments generated by restriction digestion can bereassembled in proper sequence. If restriction sites are not present atlocations within a convenient distance from the selected point (from 10to 15 nucleotides), such sites are generated by substituting nucleotidesin the gene in such a fashion that neither the reading frame nor theamino acids encoded are changed in the final construction. Mutation ofthe gene in order to change its sequence to conform to the desiredsequence is accomplished by M13 primer extension in accord withgenerally known methods. The task of locating suitable flanking regionsand evaluating the needed changes to arrive at two convenientrestriction site sequences is made routine by the redundancy of thegenetic code, a restriction enzyme map of the gene and the large numberof different restriction enzymes. Note that if a convenient flankingrestriction site is available, the above method need be used only inconnection with the flanking region which does not contain a site.

Once the naturally-occurring DNA or synthetic DNA is cloned, therestriction sites flanking the positions to be mutated are digested withthe cognate restriction enzymes and a plurality of endtermini-complementary oligonucleotide cassettes are ligated into thegene. The mutagenesis is simplified by this method because all of theoligonucleotides can be synthesized so as to have the same restrictionsites, and no synthetic linkers are necessary to create the restrictionsites.

The variant proteases expressed upon transformation of the suitablehosts can be screened for enzymes isolated or recovered exhibitingdesired characteristics, e.g. improved wash performance, substratespecificity, oxidation stability, pH-activity profiles and the like.

As used herein, proteolytic activity is defined as the rate ofhydrolysis of peptide bonds per milligram of active enzyme. Many wellknown procedures exist for measuring proteolytic activity (K. M. Kalisz,“Microbial Proteinases,” Advances in BiochemicalEngineering/Biotechnology, A. Fiechter ed., 1988). Other exemplarymethods for determining proteolytic activity include the variousspectrophotometric assays measuring the conversion of selectedsubstrates indirectly by measuring the change in absorption by theprotease added to a predetermined concentration of substrate. Exemplarysubstrates include dimethyl casein, succinyl-Ala-Ala-Pro-Phe-pNA, andkeratin (See U.S. Patent Ser. No. 60/344,702).

In addition to or as an alternative to modified proteolytic activity,the variant enzymes of the present invention may have other modifiedproperties such as K_(m), k_(cat), k_(cat)/K_(m) ratio and/or modifiedsubstrate specificity and/or modified pH activity profile. These enzymescan be tailored for the particular substrate which is anticipated to bepresent, for example, in the preparation of peptides or for hydrolyticprocesses such as laundry uses.

A change in substrate specificity can be defined as a difference betweenthe k_(cat)/Km ratio of the precursor enzyme and that of the mutant. Thek_(cat)/K_(m) ratio is a measure of catalytic efficiency. Procaryoticcarbonyl hydrolases with increased or diminished k_(cat)/K_(m) ratiosare described in the examples. Generally, the objective will be tosecure a mutant having a greater (numerically larger) k_(cat)/K_(m)ratio for a given substrate, thereby enabling the use of the enzyme tomore efficiently act on a target substrate. An increase in k_(cat)/K_(m)ratio for one substrate may be is accompanied by a reduction ink_(cat)/K_(m) ratio for another substrate. This is a shift in substratespecificity, and mutants exhibiting such shifts have utility where theprecursors are undesirable, e.g. to prevent undesired hydrolysis of aparticular substrate in an admixture of substrates.

k_(cat) and K_(m) can be measured in accord with known procedures, or asdescribed in Example 18 of U.S. Pat. No. 5,441,882.

Oxidation stability is a further objective which could be accomplishedby protease variant described in the examples. The stability may beenhanced or diminished as is desired for various uses. Enhancedstability could be effected by deleting one or more methionine,tryptophan, cysteine or lysine residues and, optionally, substitutinganother amino acid residue not one of methionine, tryptophan, cysteineor lysine. The opposite substitutions result in diminished oxidationstability. The substituted residue could be alanyl, but neutral residuesalso are suitable.

Stability, for example thermostability, is a further objective whichcould be accomplished by the protease variant described in the examples.The stability may be enhanced or diminished as is desired for varioususes. Enhanced stability could be effected by substitution one or moreresidues identified in the present application and, optionally,substituting another amino acid residue not one of the same.Thermostability is maintaining enzymatic activity over time at a giventemperature. An improved thermostability involves the maintenance of agreater amount of enzymatic activity by the variant as compared to theprecursor protease. For example an increased level of enzymatic activityof the variant as compared to the precursor at a given temperature,typically the operation temperature of as measured.

Protease variants described herein could exhibit improved washperformance under specified wash conditions. For example, the proteasevariants could exhibit differing wash performance under different washconditions, e.g. temperature, water hardness and/or detergentconcentrations as indicated by the performance determined by variousassays known in the art, e.g. WO 99/34011 (“An Improved Method ofAssaying for a Preferred enzyme and/or Preferred Detergentcomposition.”, published 8 Jul. 1999).

In the case of Bacillus subtilisin or its pre, prepro and pro forms,mutations at the earlier described positions produce mutants havingchanges in the characteristics described above or in the processing ofthe enzyme. Note that these amino acid position numbers are thoseassigned to B. amyloliquefaciens subtilisin as seen from FIG. 1. Itshould be understood that a deletion or insertion in the N-terminaldirection from a given position will shift the relative amino acidpositions so that a residue will not occupy its original or wild typenumerical position. Also, allelic differences and the variation amongvarious procaryotic species will result in positions shifts, so thatposition 169 in such subtilisins will not be occupied by glycine. Insuch cases the new positions for glycine will be considered equivalentto and embraced within the designation glycine+169. The new position forglycine+169 is readily identified by scanning the subtilisin in questionfor a region homologous to glycine+169 in FIG. 1.

One or more, ordinarily up to about 10, amino acid residues may bemutated. However, there is no limit to the number of mutations that areto be made aside from commercial practicality.

The enzymes herein may be obtained as salts. It is clear that theionization state of a protein will be dependent on the pH of thesurrounding medium, if it is in solution, or of the solution from whichit is prepared, if it is in solid form. Acidic proteins are commonlyprepared as, for example, the ammonium, sodium, or potassium salts,basic proteins as the chlorides, sulfates, or phosphates. Accordingly,the present application includes both electrically neutral and saltforms of the designated variant proteases, and the term protease refersto the organic structural backbone regardless of ionization state.

The protease variants are particularly useful in the food processing andcleaning arts. The carbonyl hydrolases, including protease variants andprecursor proteases, are produced by fermentation as described hereinand recovered by suitable techniques. See for example K. Anstrup, 1974,Industrial Aspects of Biochemistry, ed. B Spencer pp 23-46.

In one aspect of the invention, the objective is to secure a variantprotease having altered, preferably improved wash performance ascompared to a precursor protease in at least one detergent formulationand or under at least one set of wash conditions. They are formulatedwith detergents or other surfactants in accord with methods known per sefor use in industrial processes, especially laundry. In the latter casethe enzymes are combined with detergents, builders, bleach and/orfluorescent whitening agents as is known in the art for proteolyticenzymes. Suitable detergents include linear alkyl benzene sulfonates,alkyl ethoxylated sulfate, sulfated linear alcohol or ethoxylated linearalcohol. The compositions may be formulated in granular or liquid form.See for example U.S. Pat. Nos. 3,623,957; 4,404,128; 4,381,247;4,404,115; 4,318,818; 4,261,868; 4,242,219; 4,142,999; 4,111,855;4,011,169; 4,090,973; 3,985,686; 3,790,482; 3,749,671; 3,560,392;3,558,498; and 3,557,002.

There is a variety of wash conditions including varying detergentformulations, wash water volume, wash water temperature and length ofwash time that a protease variant might be exposed to. For example,detergent formulations used in different areas have differentconcentrations of their relevant components present in the wash water.For example, a European detergent typically has about 3000-8000 ppm ofdetergent components in the wash water while a Japanese detergenttypically has less than 800, for example 667 ppm of detergent componentsin the wash water. In North America, particularly the United States, adetergent typically has about 800 to 2000 ppm, for example 975 ppm, ofdetergent components present in the wash water.

A low detergent concentration system includes detergents where less thanabout 800 ppm of detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of detergent components are present in thewash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water. Brazil typicallyhas approximately 1500 ppm of detergent components present in the washwater.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 3000-8000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Asmentioned above, Brazil typically has approximately 1500 ppm ofdetergent components present in the wash water. However, other high sudsphosphate builder detergent geographies, not limited to other LatinAmerican countries, may have high detergent concentration systems up toabout 6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations ofdetergent compositions in typical wash solutions throughout the worldvaries from less than about 800 ppm of detergent composition (“lowdetergent concentration geographies”), for example about 667 ppm inJapan, to between about 800 ppm to about 2000 ppm (“medium detergentconcentration geographies”), for example about 975 ppm in U.S., andabout 1500 ppm in Brazil, to greater than about 2000 ppm (“highdetergent concentration geographies”), for example about 4500 ppm toabout 5000 ppm in Europe and about 6000 ppm in high suds phosphatebuilder geographies.

The concentrations of the typical wash solutions are determinedempirically. For example, in the U.S., a typical washing machine holds avolume of about 64.4 L of wash solution. Accordingly, in order to obtaina concentration of about 975 ppm of detergent within the wash solutionabout 62.79 g of detergent composition must be added to the 64.4 L ofwash solution. This amount is the typical amount measured into the washwater by the consumer using the measuring cup provided with thedetergent.

As a further example, different geographies use different washtemperatures. The temperature of the wash water in Japan is typicallyless than that used in Europe. For example, the temperature in Europeanwash water is generally on the order of 30 to 50 degrees centigrade,typically about 40 degrees centigrade. The temperature in North Americanand/or Japanese wash water is generally less than European wash water,for example on the order of 10 to 30 degrees centigrade, typically about20 degrees centigrade.

As a further example, different geographies use water hardness. Waterhardness is typically described as grains per gallon mixed Ca²⁺/Mg²⁺.Hardness is a measure of the amount of calcium (Ca²⁺) and magnesium(Mg²⁺) in the water. Most water in the United States is hard, but thedegree of hardness varies. Moderately hard (60-120 ppm) to hard (121-181ppm) water has 60 to 181 parts per million [parts per million convertedto grains per U.S. gallon is ppm # divided by 17.1 equals grains pergallon] of hardness minerals.

Water Grains per gallon Parts per million Soft less than 1.0 less than17 Slightly hard 1.0 to 3.5  17 to 60 Moderately hard 3.5 to 7.0  60 to120 Hard 7.0 to 10.5 120 to 180 Very hard greater than 10.5 greater than180European water hardness is typically 10-20 grains per gallon mixedCa²⁺/Mg²⁺ for example about 15 grains per gallon mixed Ca²⁺/Mg²⁺. NorthAmerican water hardness is typically greater than Japanese waterhardness, but less than European water hardness, for example, between 3to 10 grains, 3-8 grains or about 6 grains. Japanese water hardness istypically the lower than North American water hardness, typically lessthan 4, for example 3 grains per gallon mixed Ca²⁺/Mg²⁺.

Accordingly one aspect of the present invention includes a proteasevariant that shows improved wash performance in at least one set of washconditions. Another aspect of the present invention includes a proteasevariant that shows improved wash performance in more than one washconditions, e.g. in European, Japanese or North American conditions.

Based on the screening results obtained with the variant proteases, thenoted mutations in Bacillus subtilisin are important to the proteolyticactivity, performance and/or stability of these enzymes and the cleaningor wash performance of such variant enzymes.

Many of the protease variants of the invention are useful in formulatingvarious detergent compositions or personal care formulations such asshampoos or lotions. A number of known compounds are suitablesurfactants useful in compositions comprising the protease mutants ofthe invention. These include nonionic, anionic, cationic, orzwitterionic detergents, as disclosed in U.S. Pat. No. 4,404,128 toBarry J. Anderson and U.S. Pat. No. 4,261,868 to Jiri Flora, et al. Asuitable detergent formulation is that described in Example 7 of U.S.Pat. No. 5,204,015 (previously incorporated by reference). The art isfamiliar with the different formulations which can be used as cleaningcompositions. In addition to typical cleaning compositions, it isreadily understood that the protease variants of the present inventionmay be used for any purpose that native or wild-type proteases are used.Thus, these variants can be used, for example, in bar or liquid soapapplications, dishcare formulations, contact lens cleaning solutions orproducts, peptide hydrolysis, waste treatment, textile applications, asfusion-cleavage enzymes in protein production, etc. The variants of thepresent invention may comprise enhanced performance in a detergentcomposition (as compared to the precursor). As used herein, enhancedperformance in a detergent is defined as increasing cleaning of certainenzyme sensitive stains such as grass or blood, as determined by usualevaluation after a standard wash cycle.

Proteases of the invention can be formulated into known powdered andliquid detergents having pH between 6.5 and 12.0 at levels of about 0.01to about 5% (preferably 0.1% to 0.5%) by weight. These detergentcleaning compositions can also include other enzymes such as knownproteases, amylases, cellulases, lipases or endoglycosidases, as well asbuilders and stabilizers.

The addition of proteases of the invention to conventional cleaningcompositions does not create any special use limitation. In other words,any temperature and pH suitable for the detergent is also suitable forthe present compositions as long as the pH is within the above range,and the temperature is below the described protease's denaturingtemperature. In addition, proteases of the invention can be used in acleaning composition without detergents, again either alone or incombination with builders and stabilizers.

The present invention also relates to cleaning compositions containingthe protease variants of the invention. The cleaning compositions mayadditionally contain additives which are commonly used in cleaningcompositions. These can be selected from, but not limited to, bleaches,surfactants, builders, enzymes and bleach catalysts. It would be readilyapparent to one of ordinary skill in the art what additives are suitablefor inclusion into the compositions. The list provided herein is by nomeans exhaustive and should be only taken as examples of suitableadditives. It will also be readily apparent to one of ordinary skill inthe art to only use those additives which are compatible with theenzymes and other components in the composition, for example,surfactant.

When present, the amount of additive present in the cleaning compositionis from about 0.01% to about 99.9%, preferably about 1% to about 95%,more preferably about 1% to about 80%.

The variant proteases of the present invention can be included in animalfeed such as part of animal feed additives as described in, for example,U.S. Pat. No. 5,612,055; U.S. Pat. No. 5,314,692, and U.S. Pat. No.5,147,642.

One aspect of the invention is a composition for the treatment of atextile that includes variant proteases of the present invention. Thecomposition can be used to treat for example silk or wool as describedin publications such as RE 216,034; EP 134,267; U.S. Pat. No. 4,533,359;and EP 344,259.

The following is presented by way of example and is not to be construedas a limitation to the scope of the claims.

All publications and patents referenced herein are hereby incorporatedby reference in their entirety.

EXAMPLE 1

A large number of protease variants can be produced and purified usingmethods well known in the art. Mutations can be made in Bacillusamyloliqefaciens (BPN′) subtilisin or Bacillus lentus GG36 subtilisin.The variants can be selected from the following:

K27A, K27C, K27E, K27Q, K27G, K27H, K27I, K27L, K27M, K27W, K27P, K27S,K27T, K27W, K27Y, H39A, H39R, H39D, H39N, H39C, H39E, H39Q, H39G, H39I,H39L, H39K, H39M, H39S, H39P, H39T, H39W, H39Y, H39V, D41A, D41R, D41C,D41E, D41Q, D41G, D41H, D41I, D41L, D41K, D41M, D41F, D41P, D41S, D41T,D41W, D41Y, D41V, R45A, R45D, R45N, R45C, R45E, R45Q, R45G, R45H, R45I,R45L, R45K, R45M, R45W, R45P, R45S, P45I, R45W, R45Y, R45V, H67A, H67R,H67D, H67N, H67C, H67E, H67Q, H67G, H67I, H67L, H67K, H67M, H67F, H67P,H67S, H67T, H67W, H67Y, H67V, K94A, K94R, K94D, K94N, K94C, K94E, K94Q,K94G, K94H, K94I, K94L, K94M, K94F, K94P, K94S, K94T, K94W, K94Y, K94V,E136A, E136D, E136N, E136C, E136G, E136H, E136I, E136L, E136K E136M,E136W, E136P, E136S, E136T, E136W, E136Y, E136V, R170A, R170D, R170N,R170C, R170E, R170Q, R170G, R170H, R170I, R170L, R170K, R170M, R170F,R170P, R170S, R170T, R170W, R170Y R170V, D181A, D181R, D181N, D181C,D181E, D181Q, D181G, D181H, D1811, D18I, L, D181K, D181M, D181F, D181P,D181S, D0181T, D181W, D181Y, D181V, D197A, D197R, D197N, D197C, D197E,D197Q, D197G, D197H, D197I, D197L, D197K, D197M, D0197F, D197P, D197S,D197T, D197W, D197Y, D197V, R247A, R247D, R247N, R247C, R247E, R247Q,R247G, R247H, R247I, R247L, R247K, R247M, R247F, R247P, R247S, R247T,R247W, R247Y, R247V, H249A, H249R, H249D, H249N, H249C, H249E, H249Q,H249G, H249I, H249K, H249M, H249F, H249P, H249S, H249T, H249W, H249V,K251A, K251D, K251C, K251Q, K251G, K251H, K251I, K251L, K251M, K251F,K251P, K251S, K251T, K251W, K251Y, K251V, E271A, E271R, E271D, E271N,E271C, E271H, E271I, E271L, E271K, E271M, E271F, E271P, E271S, E271T,E271W, E271Y, and/or E271V of Bacillus amyloliquefaciens.

EXAMPLE 2

A large number of protease variants can be produced and purified usingmethods well known in the art. Mutations can be made in Bacillusamyoliqefaciens (BPN′) subtilisin or Bacillus lentus GG36 subtilisin.The variants can be made with insertions, deletions or substitutions atthe amino acids equivalent to those at positions: 5, 7, 23, 26, 28-31,34, 47, 63, 65, 66, 69, 70, 73, 82-85, 88, 90, 92, 93, 105, 113, 125,138, 139, 148-151, 176, 178, 179, 193, 196, 200, 201, 202, 207, 219,220, 223, 229, 233, 250, 266, 267 and 273 of Bacillus amyloliquefaciens(BPN′).

EXAMPLE 3

A large number of the protease variants produced in Examples 1 and/or 2can be tested for performance in two types of detergent and washconditions using a microswatch assay described in “An improved method ofassaying for a preferred enzyme and/or preferred detergent composition”,U.S. Ser. No. 60/068,796.

The variant proteases can be assayed and tested various detergents. Forexample, a possible detergent can be 0.67 g/l filtered Ariel Ultra(Procter & Gamble, Cincinnati, Ohio, USA), in a solution containing 3grains per gallon mixed Ca²⁺/Mg²⁺ hardness, and 0.3 ppm enzyme used ineach well at 20° C. Another exemplary detergent can be 3.38 g/l filteredAriel Futur (Procter & Gamble, Cincinnati, Ohio, USA), in a solutioncontaining 15 grains per gallon mixed Ca²⁺/Mg²⁺ hardness, and 0.3 ppmenzyme used in each well at 40° C. A higher relative value as comparedto the wild-type could indicate and improve detergent efficacy.

EXAMPLE 4

The variant proteases which can be assayed as described in Examples 1 &2 can also be assayed in other different detergents. The sameperformance tests as in Example 2 can be done on the noted variantproteases with the following detergents: a first detergent can be 0.67g/l filtered Ariel Ultra (Procter & Gamble, Cincinnati, Ohio, USA), in asolution containing 3 grains per gallon mixed Ca²⁺/Mg²⁺ hardness, and0.3 ppm enzyme could be used in each well at 20° C. A second detergentcan be 3.38 g/l filtered Ariel Futur (Procter & Gamble, Cincinnati,Ohio, USA), in a solution containing 15 grains per gallon mixedCa²⁺/Mg²⁺ hardness, and 0.3 ppm enzyme can be used in each well at 40°C. A third detergent can be 3.5 g/l HSP1 detergent (Procter & Gamble,Cincinnati, Ohio, USA), in a solution containing 8 grains per gallonmixed Ca²⁺/Mg²⁺ hardness, an 0.3 ppm enzyme can be used in each well at20° C. A fourth detergent can be 1.5 ml/l Tide KT detergent (Procter &Gamble, Cincinnati, Ohio, USA), in a solution containing 3 grains pergallon mixed Ca²⁺/Mg²⁺ hardness, and 0.3 ppm enzyme can be used in eachwell at 20° C.

EXAMPLE 5

A large number of protease variants were produced and purified usingmethods well known in the art. All mutations were made in Bacilluslentus GG36 subtilisin. The variants are shown in Table 3.

To construct the GG36 site saturated libraries and site specificvariants, three PCR reactions were performed: two PCR's to introduce themutated codon of interest in GG36 and a fusion PCR to construct theexpression vector including the desired mutation(s).

The GG36 codons of interest are numbered according to the BPN′ numbering(listed in FIGS. 1A-C and 3A-B),

For the Site Saturated Library Construction:

The method of mutagenesis was based on the region-specific mutationapproach (Teplyakov et al., 1992) in which the creation of all possiblemutations at a time in a specific DNA codon was performed using aforward and reversed complimentary oligonucleotide primer set with alength of 30-40 nucleotides enclosing a specific designed triple DNAsequence NNS ((A,C,T or G), (A,C,T or G), (C or G)) that correspond withthe sequence of the codon to be mutated and guarantees randomlyincorporation of nucleotides at that codon.

For the Site Specific Variant Construction:

The forward and reverse mutagenic primer enclose the three desiredmutation(s) in the middle of the primer with ˜15 bases of homologuessequence on both sides. These mutation(s), which cover the codon ofinterest, are specific for the desired amino acid and are synthesized bydesign.

The second primer set used to construct the libraries and variantscontains the pVS08 ApaI digestion site together with its flankingnucleotide sequence.

ApaI primers:

Forward Apal primer: GTGTGTGGGCCCATCAGTCTGACGACC Reverse Apal primer:GTGTGTGGGCCCTATTCGGATATTGAG

The introduction of the mutation(s) in GG36 molecules was performedusing Invitrogen Platinum® Taq DNA Polymerase High Fidelity (Carlsbad,Calif., Cat. no. 11304-102) together with pVS08 template DNA and Forwardmutagenic is primer and Reverse ApaI primer for reaction 1, or Reversemutagenic primer and Forward ApaI primer for reaction 2.

The construction of the expression vector including the desiredmutation(s) was accomplished by a fusion PCR using PCR fragment of bothreaction 1 and 2, forward and reverse ApaI primer and InvitrogenPlatinum® Taq DNA Polymerase High Fidelity (Cat. no. 11304-102).

All PCR's were executed according to Invitrogen protocol supplied withthe polymerases, except for the number of cycles: 20 instead of 30. Twoseparate PCR reactions are performed using Invitrogen Platinum® Taq DNAPolymerase High Fidelity (Cat. no. 11304-102): the amplified linear 5.6Kb fragment was purified (using Qiagen® Qiaquick PCR purification kitCat. no. 28106) and digested with ApaI restriction enzyme to createcohesive ends on both sides of the fusion fragment:

35 μL purified DNA fragment 4 μL React® 4 buffer (Invitrogen®: 20 mMTris-HCl, 5 mM MgCl₂, 50 mM KCl, pH 7.4) 1 μL ApaI, 10 units/ml(Invitrogen® Cat. no. 15440-019)

Reaction conditions: 1 hour, 30° C.

An additional digestion with Invitrogen Dpnl was performed to remove thepVS08 template DNA:

40 μL ApaI digested DNA fragment 1 μL Dpnl, 4 units/μL (Invitrogen® Cat.no. 15242-019)

Reaction conditions: 16-20 hours, 37° C.

Ligation of the double digested and purified fragment results in newcircular DNA containing the desired mutation with was directlytransformed to competent Bacillus subtilis:

30 μL of purified ApaI and Dpnl digested DNA fragment 8 μL T4 DNA Ligasebuffer (Invitrogen® Cat. no. 46300-018) 1 μL T4 DNA Ligase, 1 unit/μL(Invitrogen® Cat. no. 15224-017) Reaction conditions 16-20 hours, 16° C.

Ligation mixtures were transformed to Bacillus subtilis BG2864 (Naki etal., 1997) using the method of Anagnostopoulos and Spizizen (1961) andselected for chloramphenicol resistance and protease activity.

Method for Protein Production

Inoculate 1-50 μL of glycerol culture in Mops media (Frederick C.Neidhardt et al., 1974) containing carbon source (Glucose andMaltodextrine, 10.5 and 17.5 gl) a nitrogen source (Urea, 3.6 g/l), andessential nutrients such as phosphate (0.5 g/l) and sulphate (0.5 g/l)and further supplemented with trace elements (Fe, Mn, Zn, Cu; Co, 1-4mg/ml). The medium was buffered with a MOPS/Tricine mixture resulting ina pH varying 7 to 8. Incubate the culture for 1-5 days at 37° C./220 rpm(Infors HT® Multitron II).

REFERENCES

-   Protein engineering of the high-alkaline serine protease PB92 from    Bacillus alcalophilus: functional and structural consequences of    mutation at the S4 substrate binding pocket.-   Teplyakov A V, van der Laan J M, Lammers A A Kelders H. Kalk K H,    Misset O, Mulleners L J Dijkstra B W.-   Protein Eng. 1992 July; 5(5):413-20.-   Selection of a subtilisin-hyper producing Bacillus in a highly    structured environment by D. Naki. C. Paech, G. Ganshaw, V.    Schellenberger in Appl Microbiol Biotechnol (1998) 49:290-294-   Requirements for transformation in Bacillus subtilis by    Anagnostopoulos, C. and Spizizen, J. in J. Bacteriol. 81, 741-746    (1961).-   Culture Medium for Enterobacteria by Frederick C. Neidhardt,    Philip L. Bloch and David F. Smith in Journal of Bacteriology,    September 1974. p736-747 Vol. 119. No. 3.

TABLE 3 GG36 R45N G118E E271R R45N P14R R45N N204R D181N G118D R45NG258R R170S N204R R45N S216R R170S P14R R170S G61R R170S S49R R170SS216R R170S S128R R170S G258R R170S A1R R170S G100R R45N S128R R45N G61RR45N A1R D181N G258D E271T S49E E271T T66E E271T G102E E271T G100E E271TS128E K27T G100E K251G S87K

EXAMPLE 6

A large number of the protease variants produced in Example 1 weretested for performance in two types of detergent and wash conditionsusing a microswatch assay described in “An improved method of assayingfor a preferred enzyme and/or preferred detergent composition”, U.S.Ser. No. 09/554,992 [WO 99/34011].

Table 4 lists the variant proteases assayed and the results of testingin two different detergents. For column A and C, the assayed materialwas produced by growing the transformant strains in a MTP [what do theseinitials stand for?]-plate according to [citation?]. For columns B andD, the assayed material was produced by growing the transformant strainsin a shake flask (250 ml) according to [citation?]. For columns A and B,the detergent was 7.6 g/l filtered Ariel Regular (Procter & Gamble,Cincinnati, Ohio, USA), in a solution containing 15 grains per gallonmixed Ca²⁺/Mg²⁺ hardness, and 0.5 ppm enzyme was used in each well at40° C. [European conditions]. For columns C and D, the detergent was0.67 g/l filtered Tide Opal (Procter & Gamble, Cincinnati, Ohio, USA),in a solution containing 3 grains per gallon mixed Ca²⁺/Mg²⁺ hardness,and 0.5 ppm enzyme was used in each well at 20° C. [Japaneseconditions]. A performance index was calculated by the followingformula:

Cleaning performance of the variant divided by cleaning performance ofGG36 (wild-type) Four performance values were averaged to arrive at thevalues shown in Table 4.

TABLE 4 GG36 1.00 R170S-A1R 2.09 R170S-G61R 2.03 R170S-N204R 1.79R45N-G118E-E271R 1.75 D181N-G118D 1.54 R45N-N204R 1.47 K251G-S87K 1.39R45N-P14R 1.28 R45N-G258R 1.23 R170S-S216R 1.21 R45N-S216R 1.05R170S-P14R 1.03 R45N-A1R 1.01 R170S-S49R 0.93 R45N-G61R 0.87 D181N-G253D0.81 R45N-S128R 0.80 R170S-S128R 0.63 R170S-G258R 0.36 E271T-S49E 0.34E271T-G100E * E271T-T66E * E271T-G102E * R170S-G100R * E271T-S128E *K271-G100E * *too low protease level for reliable performance test

TABLE 5 variant Performance index GG36 1.00 E271T-G100E 3.22 E271T-S12SE2.33 K251G-S87K 2.06 K27T-G100E 2.04 E271T-G102E 1.85 R170S-G100R 1.79R170S-A1R 1.55 E271T-S49E 1.43 R170S-S128R 0.85 R170S-S49R 0.80R170S-N204R 0.77 R170S-P14R 0.76 R45N-A1R 0.75 R170S-G61R 0.67R170S-S216R 0.61 R45N-P14R 0.53 R45N-G258R 0.53 R170S-G258R 0.45R4SN-G61R 0.43 R45N-S216R 0.32 R45N-N204R 0.31 D181N-G258D 0.28D181N-G118D 0.28 R45N-G118E-E271R 0.25 R45N-S128R 0.22 E271T-T66E * *toolow protease level for reliable performance test ¹GG 36 is the wild typeprotease of Bacillus lentus (SEQ ID NO. 4)

As a result of the above described assays, some variants exhibited aperformance index greater than that of the GG36 wild type protease. Forexample, the variants R45N-G118E-E271R, R45N-P14R, R45N-N₂₀₄R,D181N-G118D and R45N-G258R exhibited performance indices of 1.75, 1.28,1.28, 1.24 and 1.23 respectively (Table 4), in a microswatch assay (WO99/34011) under European conditions (15 grains per gallon mixedCa²⁺/Mg²⁺ hardness, 40 degrees Centigrade, 0.5 ppm). The variantsR170S-A1P, R170S-G61R, R170S-N₂₀₄R, K251G-S87K, and R170S-S216Rexhibited performance indices of 2.09, 2.03, 1.79, 1.54, 1.47, 1.39, and1.21 respectively (Table 5). The variants E271T-G100E, E271T-G102E,E271T-S128E, K27T-G100E, R170S-G100R, and E271T-S49E exhibitedperformance indices of 3.22, 1.85, 2.33, 2.04, 1.79 and 1.43respectively (Column Table 5), in the Microswatch 96 microtiter wellplate (WO 99/34011) assay under Japanese conditions (3 grains per gallonmixed Ca²⁺/Mg²⁺ hardness, 20 degrees centigrade, 0.5 ppm). The variantsK251G-S87K, R170S-A1R, and E271T-S128E exhibited performance indices of2.06, 1.55 and 1.20 respectively (Table 5), Variants K251G-S87K andR170S-A1R exhibited performance indices of greater than 100 under bothJapanese and European conditions.

Although the present invention has been discussed and exemplified inconnection with various specific embodiments thereof, this is not to beconstrued as a limitation to the applicability and scope of thedisclosure, which extends to all combinations and subcombinations offeatures mentioned and described in the foregoing as well as theattached claims.

1-10. (canceled)
 11. A subtilisin variant of a precursor subtilisin,said subtilisin variant comprising one or more modifications at acharged amino acid residue position, in the sequence set forth in SEQ IDNO:3, said subtilisin variant being characterized by having the same netelectrostatic charge as said precursor subtilisin, wherein saidsubtilisin variant comprises an amino acid sequence having asubstitution at the residue position equivalent to residue position 251,and further comprising a substitution at one or more positionscorresponding to 1, 14, 49, 61, 87, 100, 102, 118, 128, 204, and 258.12. The subtilisin variant of claim 11, further comprising an additionalsubstitution at position corresponding to position 1, of Bacillusamyloliquefaciens subtilisin as set forth in SEQ ID NO:3.
 13. A DNAencoding the protease variant of claim
 11. 14. An expression vectorcomprising the DNA of claim
 13. 15. A host cell comprising theexpression vector of claim
 14. 16. A cleaning composition comprising theprotease variant of claim 11.